EA029862B1 - Texturizing lactic acid bacteria strains - Google Patents

Abstract

The present invention relates to mutants of lactic acid bacteria which are resistant towards the antibiotic D-cycloserine and/or functionally equivalent antibiotics and which were found to give an increased texture when grown in milk while maintaining the other growth properties of the parent strain. The present invention, furthermore, relates to compositions comprising such mutants, and to dairy products fermented with the lactic acid bacteria resistant towards D-cycloserine and/or functionally equivalent antibiotics.

Description

The invention relates to mutants of lactic acid bacteria that are resistant to the antibiotic Ώ-cycloserine and / or functionally equivalent antibiotics and which have an increased texture during growth in milk while maintaining other growth properties of the parent strain. In addition, the present invention relates to compositions that contain such mutants, and to dairy products, fermented by lactic acid bacteria, resistant to α-cycloserine and / or functionally equivalent antibiotics.

029862 Β1

029862

The technical field to which the invention relates.

The present invention relates to mutants of lactic acid bacteria, such as LascoSaicHelliski (subspecies of Schaptersch) and §1gerotrososi8 1GTOR118, which are resistant to the antibiotic ери-cycloserine and / or functionally equivalent to the antibiotics and in which there is a change. while maintaining other growth properties of the parent strain. In addition, the present invention relates to cultures, such as starter cultures, which contain such mutants, and dairy products that are fermented by these cultures.

The prior art to which the invention relates

The food industry uses numerous types of bacteria, in particular lactic acid bacteria, to improve the taste and texture of food products, as well as increase the shelf life of these food products. In the case of the dairy industry, lactic acid bacteria are intensively used to make milk souring (by fermentation), as well as for texturing the product into which these bacteria are included.

Among the lactic acid bacteria used in the food industry, the following genera can be mentioned: streptococci (гер1ger1osossi8), lactococci (Lascosossic), lactobacilli (Lascomascic), leikonostoki (Lisobasociosis). pediococci (Reyusossik) and bifidobacteria (Vysiokeksht). Lactic acid bacteria of the type 81gerotussi8 1gtorki8 are widely used individually or in combination with other bacteria, such as Las Vegaschik je1bgeski (subspecies Siberia adapci8), for the production of foodstuffs, in particular fermented products. These bacteria include, in particular, in the composition of enzymes used to produce fermented dairy products, such as yogurt. Certain bacteria among them play a leading role in the development of the texture of the fermented product. This characteristic is closely related to the production of polysaccharides.

Current trends for yogurt are moderate flavor and high texture. This is currently achieved through the use of cultures that create a mild flavor, add thickeners or proteins that give the desired consistency. Yogurt makers need the ability to make yogurt that has these properties without adding thickening agents. This will allow them to reduce costs and simplify the description of the product on the label. One very attractive way to achieve this goal is to use a starter culture that produces a high level of texture.

In order to satisfy industry requirements, it became necessary to create new texturizing strains of lactic acid bacteria, in particular, Las Vegas Syndromes (a subspecies “Biopen 8”) and § 1 Gerososssi8 (Kegtruck, for texturing food products. Especially, new texturing Vera Syros Shik Ye temsTechTyZTyyTsTyTsTyTsTyTsTyTsTyy YsTyy Ys ys Тyyyers tsеsеs. can be used together with new texturing strains of §1ger1osossi81 kegtorS1i8.

Mutants resistant to α-cycloserine have been described for a number of different bacteria, but it has never been reported that these mutants can be used to increase the texture of the dairy product. Mutants of neither 1gr1osossi81kgtorY1i8 nor laskojasShik je1icheski (subspecies bj1dapsi8) were not described.

Summary of Invention

The authors of the present invention unexpectedly found that the group of mutants of lactic acid bacteria, which are resistant to α-cycloserine, produces higher values of shear stress and / or gel stiffness than the mother strain, when these bacteria are used to ferment milk.

The authors of the present invention have proposed a method of obtaining such texturing strains of lactic acid bacteria that are resistant to the antibiotic Cycloserine and / or functionally equivalent antibiotics, and they also proposed a new group of resistant to the цикл-cycloserine strains of lactic acid bacteria of the LakosChik species. (subspecies cipher8) and §1ger1osossi8 (kegtorkkik with high texturing properties.

In accordance with the aforementioned unexpectedly discovered facts, the present invention relates to texturizing strains of lactic acid bacteria, such as Las Vegasczygus (subspecies biennial and 8), and to strains §1grosososi8 (kegtorkikik, which are resistant to Ό-cycloserine. to a functionally equivalent antibiotic, as well as to a method for producing such strains. In addition, the present invention relates to cultures, such as starter cultures, including mutants, and milk cultures. ucts, such as fermented dairy products, fermented data cultures.

Brief Description of the Drawings

FIG. Figure 1 represents the souring profiles (pH versus time) of milk at 43 ° C for the mother strain of Lascolar (a subtype of B1 adapci8) CHNSS13995 and two cycloserine-resistant mutants CHNSS13995, called CHNSS12944 and CNHS12945.

FIG. 2 represents the shear stress (at 300 1 / s (Pa)) of full-fat milk, containing 2% skimmed milk powder, at 43 ° С for the mother strain Lascomascine, which is of the same type (subspecies b1-1,09862

dapsic) CNSS13995 and two Ό-cycloserine-resistant mutants of SNCS13995, called SNSS12944 and CNSS12945.

FIG. 3 represents the gel hardness (at 300 1 / s (Pa)) of full-fat milk containing 2% skimmed milk powder, 223 ° C for the mother strain B. and CNSS12945.

FIG. 4 represents the gel hardness (at 300 1 / s (Pa)) of full-fat milk, containing 2% skimmed milk powder, at 43 ° C for the mother strain 81ter1osossik Shettork1CHCS13994 and two цикл-cycloserine-resistant mutants CXC13994, called CNSS13235 and CCHCl336

FIG. 5 represents the gel hardness (at 300 1 / s (Pa)) of full-fat milk, containing 2% skimmed milk powder, at 43 ° C for the mother strain 81ter1osossik Shettork1CHCS13994 and two Ό-cycloserine-resistant mutants CXC13994, called CNSS13235 and CCHCl3323

Detailed description

Definitions

As used herein, the term “lactic acid bacteria” means gram-positive microaerophilic or anaerobic bacteria that ferment sugars, and this produces acids, including lactic acid, as the predominantly formed acid, acetic acid, and propionic acid. The most useful for the industry lactic acid bacteria are in the detachment of lactobacilli (lacobicus), which are the contents of the following genera: lactococci (lacobicus), streptococci (glorious and osteotic), lactobacilli (lacobacillus), lactobacilli, lactobacilli, lactobacilli (lactobacilli), lactobacilli (lacobacillus) brevibakteriya (VgePyaspups). enterococci (Eytossosik) and propionibakterii (Roryutya Lettsht). In addition, lactic acid-producing bacteria belonging to the group of strictly anaerobic bacteria, namely, species of the genus Bifidobacteria (WLTL), are usually included in the group of lactic acid bacteria. They are often used as food crops individually or in combination with other lactic acid bacteria. Lactic acid bacteria, including bacteria of the lactobacillus species (LacFacSych) and thermophilic streptococci (11ter!, ShegtorNPik), are usually supplied to the dairy industry in the form of frozen or lyophilized cultures for bulk initiation or in the form of so-called direct seeding cultures (Όνδ), which designed for direct inoculation into a fermentation tank or tank for the production of a dairy product, such as a fermented dairy product. Such cultures are referred to by the general term “starter cultures” or “initiators”.

The term “milk” should be understood as a liquid released from the mammary glands of any mammals, such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk. The term "milk" also includes solutions containing proteins and fats made from plant materials, such as soy milk.

The term “dairy substrate” can be any raw and / or processed dairy material that can be fermented using the method of the present invention. Thus, dairy substrates used include, but are not limited to, solutions / suspensions of any milk or milk-like product containing a protein, such as whole or low-fat milk, skimmed milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, low protein whey (permeate), lactose, mother liquor from lactose crystallization, whey protein concentrate or cream. Of course, the milk substrate can be obtained from any mammal, for example, it can be almost pure mammalian milk or reconstituted milk powder.

Preferably, at least a portion of the protein in the milk substrate are proteins contained in natural milk, such as casein or whey protein. However, part of the protein can be proteins that are not found in natural milk.

Before fermentation, the milk substrate can be homogenized and pasteurized using methods known in the art.

The term "homogenization" as used herein means vigorous stirring to obtain a soluble suspension or emulsion. If the homogenization is carried out before fermentation, it can be carried out in such a way as to break up the milk fat into smaller particles that are no longer separated from the milk. This can be done by passing milk at high pressure through small holes.

The term "pasteurization" as used herein means treating a dairy substrate to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is carried out by aging at a certain temperature for a certain period of time. The desired temperature is usually achieved by heating. The temperature and duration of treatment can be chosen in such a way as to destroy or inactivate certain bacteria, such as harmful bacteria. After that you can carry out the stage of rapid cooling.

The term "fermentation" in the methods of the present invention means the conversion of carbohydrates to

- 2 029862

alcohols or acids under the action of microorganisms. Preferably, the fermentation in the methods of the present invention is the conversion of lactose to lactic acid.

Fermentation processes for use in the production of fermented dairy products are well known and one skilled in the art knows how to select suitable process conditions, such as temperature, oxygen, the quantity and characteristics of microorganisms, and the duration of the process. Of course, the fermentation conditions are chosen in such a way as to ensure the achievement of the objective of the present invention, i.e. get dairy product in solid or liquid form (fermented dairy product).

In the context of the present invention, a yoghurt starter culture is a bacterial culture that is a fermented dairy product obtained by inoculation and fermentation of milk using a composition comprising a strain of Bacilobi8 bilgia (subspecies bilapadi8) and a strain of §1ger1osioci8 1gr Sy1i8.

In the context of the present invention, the term "shear stress" defines viscosity. Viscosity (expressed in Pa-s) is defined as the ratio of shear stress (Pa) to shear rate (1 / s).

The voltage value is standardly defined in this document at a shear rate of 300 1 / s.

Organoleptic experiments (data not shown) showed that the best correlation between rheological measurements and organoleptic viscosity (sensation in the oral cavity) is found when using a viscosity measured at a shear rate of 300 1 / s.

In the context of the present invention, the term "functionally equivalent antibiotic" should be understood as an antibiotic having the same mechanism of action or the same target as Ό cycloserine, such as, for example, other inhibitors of л-alanyl-O-alanine ligaz, such as, for example, vancomycin ( Tu1da1 et al., Sigg. Meb. Syet., 2009, Vol. 16, No. 20, pp. 2566-2580) and other alanine racemase inhibitors, such as, for example, O-carbamoyl-E-serine, alaphosphine, and haloalanine (No11) e Eezk Epps1oreFa GO M1SgoIy1odu (Desktop Encyclopedia of Microbiology), 2004, p. 239-258).

The term "Ό-cycloserine-resistant and / or functionally equivalent antibiotic" in this document means that a particular mutated bacterial strain is not destroyed or is destroyed much slower compared with the corresponding nemutriuyushchy strain from which the mutated strain was obtained in the presence of the indicated antibiotic in culture medium. Depending on the concentration of the antibiotic compound in the culture medium, changes in the growth properties of mutated and non-mutated strains may also affect resistance. For example, a low concentration of antibiotic in the culture medium prevents or significantly inhibits the growth of non-mutated strains without affecting the growth of mutated strains. Non-mutated strains that can be used as sensitive reference strains for the assessment of resistance preferably include strains CNSS13995 and CNSS13994.

Examples 1 and 2 of this application illustrate the isolation of mutant strains of Escalobacci8 bélbieski (subspecies biladapi8) and §1ger1oschospi81grrr118, respectively, which are resistant to-cycloserine. Based on the methods presented in examples 1 and 2, one skilled in the art can easily select for each unmutated maternal strain the amount of α-cycloserine or the amount of functionally equivalent antibiotic, which by its action destroys most cells of the maternal strain and does not destroy or destroy significantly slower mutant strains resistant to Cycloserin and / or a functionally equivalent antibiotic.

In the context of the present invention, the term "mutant" should be understood as a derived strain or strain that can be obtained from the strain according to the present invention (or the parent strain), using, for example, genetic engineering methods, radiation and / or chemical treatment. The mutant may also be a spontaneously arising mutant. It is preferred that the mutant is a functionally equivalent mutant, for example, a mutant that has almost identical or improved properties (for example, viscosity, gel hardness, oral cavity coating, aroma, subsequent souring, souring speed and / or phage resistance) compared to maternal strain. Such a mutant is a part of the present invention. In particular, the term "mutant" refers to a strain obtained by exposing a strain according to the present invention to any traditionally used mutagenic treatment, including treatment with a chemical mutagen, such as ethyl methane sulfonate (EM§) or M-methyl-N-nitro-Nnitrozoguanidine ( ΝΤΟ), or ultraviolet radiation, or to a spontaneously occurring mutant. A mutant can be subjected to several mutagenesis procedures (one procedure should be understood as one stage of mutagenesis, followed by an inspection / selection stage), but for the present invention it is preferable to perform no more than 20, or no more than 10, or no more than 5 procedures ( or verification / selection stages). In the mutant preferred for the present invention, less than 1, less than 0.1, less than 0.01, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome are replaced by other nucleotides or are removed compared to the parent strain.

- 3 029862

In the context of the present invention, the term "variant" should be understood as a strain that is functionally equivalent to the strain according to the present invention, for example, having almost the same or improved properties, such as viscosity, gel hardness, oral cavity coating, aroma, subsequent souring, speed souring and / or phage resistance. Such options that can be identified using appropriate research methods are part of the present invention.

The use of definite and indefinite articles and similar auxiliary words in the context of describing the present invention (in particular, in the context of the following claims) should be interpreted as a single designation of singular and plural, unless another condition is defined in this document, or if it is not clearly in conflict with the context. . The terms "representing", "having", "including" and "containing" should be interpreted as non-limiting terms (i.e., having the meaning "including but not limited to") unless otherwise specified. The representation of intervals of values in this document is intended simply as a way to reduce the individual listing of each individual value that is in this interval, unless a different condition is defined in this document, and each individual value is included in this description as if it were individually presented in this document. . All the methods described in this document can be carried out in any suitable order, unless a different condition is defined in this document, or if it is not clearly in conflict with the context. The use of any and all examples or corresponding expressions (eg, “such as”) in this document is intended merely to better illustrate the present invention and does not impose limitations on the scope of the present invention unless otherwise specified. None of the expressions in the present description should be interpreted as meaning that any element not claimed is essential for the practical implementation of the present invention.

The implementation and aspects of the present invention.

Ό-Cycloserine (Ό-4-amino-Z-isoxazolidone) is an antibiotic that inhibits alanine racemase, Ό-alanyl-O-alanine ligase, ла-alanylalanine synthase and Ό-alanine permease, resulting in lysis of the cells. Ό-alanine racemase plays an important role in the synthesis of-alanine, an integral part of the peptidoglycan cell wall layer. Mycobacterium mutants (Musoblaste), which are resistant to β-cycloserine, produce an excess of α-alanine by increasing the expression of the α-alanine racemase gene (Sasegek et al., Ί. Vasile1., 1997, vol.179, pp. 5046-5055 ). Thus, the authors of the present invention of the present invention stated the possibility of isolating spontaneous mutant strains of lactic acid bacteria, which are characterized by the production of a larger amount of α-alanine. Such mutants were isolated and characterized.

The person skilled in the art understands that other antibiotics having the same mechanism of action or the same target as α-cycloserine can be used individually or in combination with α-cycloserine to isolate mutants of the type described herein. Thus, the present invention also includes the use of such other functionally equivalent antibiotics, such as other α-alanyl-O-alanine ligaz inhibitors or α-alanine racemase.

The term "functionally equivalent antibiotics with respect to α-cycloserine" as used herein includes any compound that interferes with the synthesis of α-alanine, transfer of α-alanine into the cell, the formation of α-alanyl-O-alanine from α-alanine or incorporation of -alanyl-alanine in the constituent elements of the peptidoglycan polymer. Such antibiotics include, but are not limited to, antibiotics that inhibit the following enzymes: Ό-alanine-racemase, Ό-alanyl-O-alanine ligase, Ό-alanylalanine synthase and Ό-alanine permease. Such functionally equivalent antibiotics with respect to α-cycloserine include, but are not limited to, vancomycin, O-carbamoyl-O-serine, alaphosphine, and haloalanine.

It has been found that some mutants of β1β1,8S8i4e1Hgieski (subspecies bj1dagyi8) and §1ger1osssi81gtrS1i8, which are resistant to α-cycloserine, provide an increased texture in the process of growth in milk compared to the parental strain while maintaining other parental growth properties

In accordance with the above-mentioned unexpectedly discovered facts, the present invention in the first aspect relates to a method for producing a texturing strain of lactic acid bacteria, said method comprising:

a) providing a maternal strain of lactic acid bacteria;

b) isolating a mutant of the parent strain that is resistant to Ό cycloserine and / or a functionally equivalent antibiotic with respect to Ό-cycloserine; and

c) selecting a mutant if it produces a better texture, which is determined by an increase in shear stress and / or an increase in gel stiffness during growth in milk than the mother strain, resulting in a texturizing strain of lactic acid bacteria.

Thus, the first aspect relates to a method for producing a mutant lactic acid bacteria, which provides an increase in shear stress and / or gel stiffness during growth in milk,

- 4 029862

moreover, this method includes:

a) providing a maternal strain of lactic acid bacteria;

b) isolating a mutant of the parent strain that is resistant to Ό cycloserine and / or a functionally equivalent antibiotic with respect to Ό-cycloserine; and

c) selecting a mutant if it produces a better texture, which is determined by an increase in shear stress and / or an increase in gel stiffness during growth in milk than the parent strain, which results in a mutant of lactic acid bacteria that provides an increase in shear stress and / or gel stiffness during growth in milk.

The production of a larger texture by the selected mutant as compared to the maternal strains, which is determined by an increase in shear stress and / or by an increase in gel stiffness, is carried out in both full-fat and low-fat milk.

In a preferred embodiment, the milk is cow's milk. Preferably, the milk is full-fat cow milk.

In a preferred embodiment, the maternal strain of lactic acid bacteria is lactic acid bacteria selected from the group consisting of LacFBeSchic bélie (subspecies bbl1dagyi8) and 11grössosi8 1égtor X1 and 8.

However, a part of the present invention is also the use of other lactic acid bacteria, such as bacteria of the following genus types: lactococci (Lacpossi8), streptococci (§1ger1oscussi8), lactobacilli (Basilochosi8), leukostoka (Beisopocephalus). pediococci (Rebusessic), brevibacteria (Veu1us1e), enterococci (Euhessossic), propionibacteria (Protophagia!) and bifidobacteria (Vecchiose).

In one preferred embodiment, the mutants are obtained by incubating the maternal strain of lactic acid bacteria in a volume / on the surface of the growing medium containing Ό cycloserine and / or a functionally equivalent antibiotic with respect to цикл-cycloserine, and isolating the mutant of the parent strain that does not destroy- cycloserine and / or a functionally equivalent antibiotic in relation to α-cycloserine, or which is destroyed much slower than most of the neutirovanny cells studied. However, the present invention provides other ways known to the person skilled in the art in order to isolate mutants resistant to α-cycloserine and / or functionally equivalent antibiotics with respect to cycloserine. Accordingly, the mother strain used in the method according to the first aspect of the present invention is a strain that is not resistant (or is less resistant) to β-cycloserine and / or a functionally equivalent antibiotic with respect to α-cycloserine.

In one embodiment, the mutant may be a spontaneous Ό cycloserine resistant mutant and / or a functionally equivalent antibiotic with respect to цикл-cycloserine.

In yet another embodiment of the present invention, the method may include a step of mutagenesis (for example, by chemical treatment or exposure to radiation or using genetic engineering methods) of the parent strain, for example, before or during stage b).

Thus, in one embodiment, the mutant can be obtained by mutagenesis of the parent strain, using, for example, chemical treatment or radiation exposure, or the mutant can be obtained using genetic engineering techniques.

The terms "texture" or "sensation in the oral cavity" mean the chemical and physical interaction of the product in the oral cavity.

Methods for determining the texture of milk include measuring the shear stress (viscosity) or stiffness of a gel of fermented milk, and they are readily available and known in the art, and are also illustrated with examples in this document.

The mutant is selected in step c) if it produces a better texture, which is determined by an increase in shear stress and / or an increase in gel hardness, in fermented milk than the mother strain, when inoculated in the same amount as the mother strain, for example, in the amount of at least 10 ⁴ cells per ml.

In a preferred embodiment, the mutant produces a viscosity in milk, measured as a shear stress at a rate of 300 1 / s (Pa), which is at least about 5% higher, at least about 10% higher, at least about 15 % higher, at least about 20% higher, at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher, at least at least about 50% higher than viscosity l produced by the parent strain.

In another preferred embodiment, the mutant produces a gel hardness in milk that is at least about 5% higher, at least about 10% higher, at least about 15% higher, at least about 20% higher at least about 25% higher, at least about 30% higher, at least about 35% higher, at least about 40% higher,

- 5 029862

at least about 50% higher than the stiffness of the gel produced by the parent strain.

Preferably, the mutant produces viscosity in full-fat cow's milk with the addition of 2% skimmed milk powder, which is more than about 105 Pa when measured as a shear stress at a rate of 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C with inoculation into a quantity of at least 10 ⁴ cells per 1 ml of milk in the case of strains of Las beats 8 (1) (more than approximately 75 Pa, measured as a shear stress at a speed of 300 1 / s (Pa) after 12 hours of growth in milk with 37 ° C with inoculation in the amount of constituting at least 10 ⁴ cells per 1 ml of milk in the case of the strains

In a preferred embodiment, the mutant produces gel hardness in full-fat cow's milk with the addition of 2% skimmed milk powder, which is more than about 80 Pa after 12 hours of growth in milk at 37 ° C with inoculation in an amount of at least 10 ⁴ cells per 1 ml of milk in the case of the BésoBiuSi8 bébégiski strains (subspecies b1dapi8), and more than about 140 Pa after 12 h of growth in milk at 37 ° C with inoculation in an amount of at least 10 ⁴ cells per 1 ml of milk in the case of strains §! terossoss 8 1yegtorY1i8.

In a second aspect, the present invention relates to a strain of lactic acid bacteria obtained by the method according to the first aspect of the present invention.

In a preferred embodiment, the lactic acid bacterium strain is a strain selected from the group consisting of BaciloSchi8 bécie (subspecies bb1datyi8) and §1.

In another preferred embodiment, the strain of lactic acid bacteria produces viscosity in full-fat cow's milk with the addition of 2% skimmed milk powder, which is more than about 105 Pa when measured as shear at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C with inoculation in an amount of 10 ⁴ cells per 1 ml of milk in the case of strains of Bas1oSchic jer1 (subspecies bj1datui8), and more than approximately 75 Pa when measured as shear stress at a speed of 300 1 / s (Pa) after 12 h ro that in milk at 37 ° C with the inoculation in an amount of at least 10 ⁴ cells per 1 ml milk in the case of strains §! ter1osossi8! egtorY1ik.

Preferably, the lactic acid bacterial strain produces gel hardness in full-fat cow's milk with the addition of 2% skimmed milk powder, which is more than about 80 Pa after 12 hours of growth in milk at 37 ° C with an inoculation in an amount of at least 10 ⁴ cells per 1 ml of milk in the case of strains of Bacillobacterium (subspecies biblical), and more than approximately 140 Pa after 12 hours of growth in milk at 37 ° C with inoculation in an amount of at least 10 ⁴ cells per 1 ml of milk in the case of strains § 1ter1osossi8 (N itsNPik.

In the third aspect, the present invention relates to a strain of lactic acid bacteria belonging to the species Bacillus Synthesis (subspecies bipedaline 8) selected from the group consisting of: a strain of CNSS12944, which is deposited in the German Collection of Microorganisms and Cultures under the registration number ΌδΜ СΜΜJVJ1719444; which is deposited in the German collection of microorganisms and cultures under the registration number ΌδΜ 24020, and mutants and variants of any of the strains listed above. Mutants and variants exhibit characteristics of shear stress and / or stiffness of the gel of the deposited strains from which they were derived.

In the fourth aspect, the present invention relates to a strain of lactic acid bacteria belonging to the species гер! Herthosis ShettorNLik selected from the group consisting of: strain CNSS13235, which is deposited in the German Collection of Microorganisms and Cultures under registration number ΌδΜ 24010, strain CNSS13236, which is deposited in the German collection of microorganisms and cultures under the registration number ΌδΜ 24011, and mutants and variants of any of the strains listed above. Mutants and variants exhibit characteristics of shear stress and / or stiffness of the gel of the deposited strains from which they were derived.

In the fifth aspect, the present invention relates to a strain of lactic acid bacteria belonging to the species Casicobacidus (subspecies csubacidi8), resistant to Cyclocerin and / or a functionally equivalent antibiotic and having the same or improved characteristics of shear stress and / or stiffness of the gel compared to strain CNSS12944, which is deposited in the German Collection of Microorganisms and Cultures under registration number ΌδΜ 24019, or strain CNSS12945, which is deposited in the German Collection of Microorganisms Cultures under accession number 24020 ΌδΜ.

In the sixth aspect, the present invention relates to a strain of lactic acid bacteria belonging to the species §1 ter1osossi ShettorNLik resistant to α-cycloserine and / or a functionally equivalent antibiotic and having the same or improved characteristics of shear stress and / or rigidity of the gel compared to the strain CNSS13235, which is deposited in the German Collection of Microorganisms and Cultures under the registration number ΌδΜ 24010, or in strain CNSS13236, which is deposited in the German Collection of Microorganisms and cultures under the Registration number Ό δΌ 24011.

- 6 029862

In the seventh aspect, the present invention relates to a composition comprising a lactic acid bacteria strain according to aspects 2 to 6 of the present invention.

In one embodiment of the present invention, the composition comprises in the form of a mixture or components of the kit:

ί) a strain belonging to species 5> 1 ger1osossi5 Ιΐιοπηορίιίΐιΐδ according to the second, fourth, or sixth aspects of the present invention; and

) a strain belonging to the species Bosocoscidus de jethus (subspecies bipedalisus 8) according to the second, third, or fifth aspects of the present invention.

In one embodiment, the composition preferably comprises at least 10 ⁹ cfu (colony forming units) of a strain belonging to the species baciocus8 b1bgien (subspecies bb1dat1c8); and at least 10 ¹⁰ cfu of the strain belonging to species 5> Gergerososs5 ShettorShik. In yet another embodiment, the composition contains at least 10 ¹¹ cfu of a strain belonging to the bicusbicus species (subspecies biennial 8); and at least 10 ¹² cfu of the strain belonging to species 5> 1gerosossi5 LettorI1i8.

In one preferred embodiment, the composition can be advantageously used as a starter culture, and it is in frozen, lyophilized, or liquid form.

In the eighth aspect, the present invention relates to a method for producing a dairy product, such as a fermented dairy product, comprising fermenting a dairy substrate with a strain of lactic acid bacteria according to any one of aspects 2 to 6 of the present invention, or a composition according to the seventh aspect of the present invention, and as a result dairy product.

As a result, the dairy product made by this method contains a strain of lactic acid bacteria according to any one of aspects 2-6 of the present invention, or strains of lactic acid bacteria of a composition according to the seventh aspect of the present invention, respectively.

According to this method, the dairy substrate can be fermented using a strain according to the present invention, such as a strain belonging to the species 1 1ter1osossi8 1ygtor HI1 and 8 before, during or after fermentation with a strain according to the present invention, such as a strain belonging to the type 1 bas1oS8i8.

In addition, this method may include, before, during and / or after fermentation, the introduction into the milk substrate of the enzyme, substance, thickening agent, flavoring and preservative; and / or its form is optionally a mixture type product, a kit type product or a drinking product.

In a tenth aspect, the present invention relates to a dairy product comprising a strain of lactic acid bacteria according to any one of aspects 2 to 6 of the present invention.

In an eleventh aspect, the present invention relates to the use of a strain of lactic acid bacteria according to any one of aspects 2 to 6 for the production of a fermented milk product.

Further, the present invention is illustrated by the following non-limiting examples.

Examples

Example 1. The use of β-cycloserine for the isolation of mutants of Ba1: wasca8 be1bieskp (subspecies bb1dat1ci8) with improved rheological properties.

Materials and methods.

Strains.

Lac1: ABOUT 8 BER1HGESCP (subspecies Shdapsik) SNSS13995.

Lac1: ABOUTS 8 jerk (subspecies bb1Dagy8) SNSS12944 (Cyclosarine-resistant mutant CHNSS13995).

Lac1: SUSHI 8 jerk (subspecies Bj1 dagui8) CHSS12945 (Cyclosserin-resistant mutant CHNSS13995).

Isolation of mutants.

In order to isolate mutants of the Lac1 strain: CUSHI8 BESHGIESKI (subspecies B1Dagyi8) of CNSS13995, cells obtained with the growth of a single colony were inoculated in MK2 broth containing from 50 to 100 µg / ml Ό-cycloserine, and grown until saturation. Cells were diluted and plated on MKs plates, and colonies were tested in microliter wells for growth in the presence of α-cycloserine in the range from 50 to 100 μg / ml. As a rule, 25% of the resulting colonies showed rapid growth in the presence of α-cycloserine. They were chosen for further research. The selected Ό-cycloserine-resistant mutants were further purified and examined for growth in milk. During this study, it was observed that some of the resulting mutants under these conditions produced a significantly better texture than the parent strain.

Rheology.

The rheological analysis was carried out using a rheometer §18888 from the company ReoHofsPtcitepts AB (Sweden), after growing in full-fat cow's milk with the addition of 2% dry non-fat

- 7 029862

milk

Results.

Two derivatives of the mutant CHCS13995, designated SNSS12944 and CNSS12945, produced a significantly better texture, which was distinguished by increased shear stress (viscosity), as well as gel stiffness, than the maternal strain. This was quite obvious with the simple mixing of fermented milk with a pipette. In addition, it was observed that the amount of whey was different for different mutants, and it became one of the parameters used to select mutants for further research (see Table 1 below). The selected mutants were those that showed a low level of syneresis: mutant 1 and mutant 11 in the example presented below (Table 1) are mutants of CNSS12944 and CNSS12945, respectively.

Table 1

Mutant 1 and mutant 11 were selected from among 20 mutants due to the small amount of milk whey they produce. Empty columns refer to mutants that do not roll milk. Two mutants are SNSS12945 and SNSS12944 respectively

Mutant one 2 3 four five 6 7 eight 9 ten eleven 12 13 14 15 sixteen 17 18 nineteen 20 ML four sixteen - 17 eight eight 10.5 22 - sixteen 7 five 15 32 eleven - sixteen 20 - -

The volume (ml) of whey after growth for 24 hours in 100 ml of category B milk at 43 ° C. Actual rheological studies confirm observations of consistency improvement.

(tab. 2 and figs. 2 and 3). The increase in gel stiffness in the case of mutants CHCS12945 and CHCS12944 is from 52 to 59%. The shear stress also increases, but not to such a large extent as the rigidity of the gel (Table 2 and Figs. 2 and 3).

table 2

Sample Shear stress at 300 1 / s (Pa) Gel hardness at 1 Hz (Pa) CNSS13995 99 58.55 CNSS12945 108.5 93.2 0 CNSS12944 108 89.05

Improving the rheological properties is only one advantage, but mutants become less attractive if other important properties, such as souring, deteriorate. The souring profile with growth in full-fat cow's milk with the addition of 2% skimmed milk powder at 43 ° C is the same for the selected mutants and for the corresponding maternal strain (Fig. 1).

Conclusion.

The β-cycloserine-resistant mutants of lacFbc sciens b1 (subspecies b1 adaps5) described herein can be included in a culture, such as a starting culture, which produces a desirable high level of texture.

Example 2. The use of α-cycloserine for the selection of mutants ZTseorFossiss Shegshor'yiz with improved rheological properties.

Materials and methods.

Strains

ZierFsossiz ShegshorPiz SNSS13994

ZierFssossiz Shegshoryyiz SNSS13236 (mutant resistant to α-cycloserine mutant SNSS13994)

ZierFsossiz Shegshoryyiz SNSS13995) (mutant of Ό-cycloserine mutant SNSS13994) resistant

Isolation of mutants.

In order to isolate the mutants of ZCPS in order to isolate a single colony, it was inoculated in M17 broth with the addition of 2% lactose containing from 50 to 70 μg / ml cycloserine and grown to saturation. The cells were diluted and plated on M17 plates with the addition of 2% lactose, and the colonies were harvested and checked in microliter wells for growth in the presence of α-cycloserin in the range from 50 to 70 μg / ml α-cycloserine. As a rule, 25% of the resulting colonies showed rapid growth in the presence of α-cycloserine. They were chosen for further research. Selected resistant to α-cycloserine mutants were further purified and examined for growth in milk. During this study, it was observed whether some of the mutants produced a significantly better texture under the given conditions than the manufacturer’s parental strain. The amount of whey was also a measure showing high or low gel hardness.

Rheology.

Rheological analysis was carried out using a ZCEZRES rheometer from Keofofs 1p81gishep18 AB (Sweden), after growing in full-fat cow's milk with the addition of 2% dry skim milk.

- 8 029862

milk

Results.

The two derivatives of the mutant CHCS13994, designated CHCS13235 and CHCS13236, produced a better texture than the parent strain. This was quite obvious with the simple mixing of fermented milk with a pipette. In addition, it was observed that the amount of whey was different for different mutants, and it became one of the parameters used to select mutants for further research (see Table 3 below). Selected mutants were those that showed a low level of syneresis: mutant 9 and mutant 13 in the table. 3 showed a low level of syneresis. These mutants are CNSS13236 and CNSS13235, respectively.

Table 3

Two mutants (9 and 13) are selected from among 20 mutants

Mutant one 2 3 four five 6 7 eight 9 ten eleven 12 13 14 15 sixteen 17 18 nineteen 20 ml 12 20 ten 5.5 7 ten 7.5 12 7 22 65 - 65 7 75 6 ten ten five 6

The volume (ml) of whey after growth for 24 hours in 50 ml of category B milk at 37 ° C.

Actual rheological studies confirm observations of improvements in consistency (Fig. 4 and 5). The mutant CHCS13236 showed an increase in shear stress by 15% and an increase in gel stiffness by 19%, while the mutant CHCS13235 showed only an increase in gel hardness by 23% (Table 4).

Table 4

Sample Shear stress at 300 1 / s (Pa) Gel hardness at 1 Hz (Pa) CNSS13994 69,6 136.6 CNSS13236 79.7 162.5 CNSS13235 64.9 167.3

As for souring, the profile is the same for the selected mutants and for the corresponding maternal strain (data not shown).

Conclusion.

The α-cycloserine-resistant mutants of ZDerFossiss ShegzhorPiz described herein can be incorporated into a culture, such as a starting culture, which produces a desired high level of texture.

Example 3. The use of Ό-cycloserine-resistant mutants ZDFssossiz ShegzhorPiz and EASFbashShiz b1ngieskl (subspecies b1 adapsis) for the preparation of b1bieski (subspecies cb1 adapsiz) of SNSS12945.

Comparison of souring profiles for these three combinations showed that the combinations that contained two mutants had a slightly higher pH value than the combination of parental strains (data not shown). Stirring with a spoon showed that products made using mutants resistant to α-cycloserine showed a significantly higher resistance to mixing compared to the product made using parental strains. In addition, it turned out that the resulting yogurt had a thicker creamy texture and a fresher taste, according to three experts.

Depositing and expert decision.

EasFyasShiz bégieski (subspecies) csnc12944, cnsx12945 and csncrrrrrrrrrrrrrrrrrrbdhmgrcdweb.com September 22, 2010

The strains ZDerFssossiz ShegshorPiz SNSS3232, SNSS13236, and the CPC programs were deposited in the German Collection of Microorganisms and Cultures LLC (Ό3ΜΖ OshN, Germany, Ό-38124, Braunschweig, Inhofenstraße, 7B) under registration numbers, under registry numbers, under registration numbers, in Braunschweig, Inhofenstrasse, 7B year

The deposit was made in accordance with the terms of the Budapest Agreement on the International Recognition of the Deposit of Microorganisms for the purposes of granting patents.

The applicant requests to provide a sample of the deposited microorganisms only to an expert approved by the applicant.

Bibliography.

Tufa! et al., Sigg. Meb. Sat., 2009, vol. 16, No. 20, p. 2566-2580.

Sasegez et al., I. VasPhoPo1., 1997, v.179, p. 5046-5055.

11o1re, E) Eccus1oreSa o £ MügöFFFdu (Desk Encyclopedia of Microbiology), 2004, pp. 239-258.

All the literature cited in this patent document is included in its entirety by reference.

- 9 029862

Claims (14)

CLAIM 1. A method of obtaining a texturing strain of lactic acid bacteria, and this method includes: a) providing a maternal strain of lactic acid bacteria; b) isolating a mutant of the indicated maternal strain that is resistant to Ό-cycloserine and / or a functionally equivalent antibiotic, by culturing the mother strain in the presence of the indicated antibiotic, where the indicated functionally equivalent antibiotic prevents the synthesis of Ό-alanine, transfer from the culture medium Ό- alanine in the cell, the formation of α-alanyl-O-alanine from α-alanine or the inclusion of O-alanyl-O-alanine in the structural elements of the peptidoglycan polymer in the bacterial cell; and c) selection of a mutant from mutants selected in stage b), ί) if it provides the best texture, which is determined by the increase in shear stress during growth in milk than the parent strain, where the mutant is chosen if it produces a shear stress that is at least approximately 5% more than the shear stress produced by the parent strain, and where the shear stress is measured at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C, or (ίί) if it provides gel rigidity, which is at least approximately 5% more than the stiffness of the gel produced by the parent strain. 2. The method according to claim 1, wherein said mutant is selected in step c) if it produces a shear stress that is at least about 10% greater than the shear stress produced by the parent strain. 3. The method according to any one of the preceding claims, wherein said maternal strain of lactic acid bacteria is a strain selected from the group that consists of Basus and Graceless (subspecies) and HERGss. 4. A stable towards Ό-cycloserine texturizing strain of lactic acid bacteria as1oasShi8 be1gieski (subspecies i1dag1si8) SNSS12944 which is deposited under accession number Ό8ΜΖ Ό3Μ 24019, and mutant strain thereof produced by genetic engineering, by means of radiation and / or chemical treatments that generate shear stress in full-fat cow's milk with the addition of 2% skimmed milk powder more than 105 Pa when measured at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C. 5. Resistant to α-cycloserine texturizing strain of lactic acid bacteria BaCiOrSiS 8 beliBicheski (subspecies io1dagiCi8) SNSS12945, which is deposited in "3" under registration number "3" 24020, as well as the mutant strain, produced by it under the methods 3Μ 24020, as well as the mutant strain, produced by it under the methods методами 3Μ 24020, as well as the mutant strain deposited by it using the methods under the registration number 3Μ 24020, as well as the mutated strain deposited by it using the methods under the registration number 203 24020, and the mutant strain produced by it using the methods under the registration number 203Μ 24020, and the mutant strain produced from it under the registration number No. 3 2402020 or chemical treatments that generate shear stress in full-fat cow's milk with the addition of 2% skimmed milk powder more than 105 Pa when measured at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C. 6. Resistant to ос-cycloserine texturizing strain of lactic acid bacteria JQWssiSyrGy1 and 8 JHC13235, which is deposited in Ό3ΜΖ under registration number Ό3Μ 24010, as well as a mutant strain produced by methods of genetic engineering, when exposed to radiation and / or chemical treatment, which generate shear stress in full-fat cow's milk with the addition of 2% skimmed milk powder of more than 75 Pa when measured at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C. 7. Resistant to ос-cycloserine texturizing strain of lactic acid bacteria JQWssiSyrGy1 and 8, which is a CNSS13236 strain, which is deposited in Ό3ΜΖ under registration number Ό3Μ 24011, as well as a mutant strain produced from it by genetic engineering methods, when exposed to radiation / or chemical treatments that generate shear stress in full-fat cow's milk with the addition of 2% skimmed milk powder more than 75 Pa when measured at 300 1 / s (Pa) after 12 hours of growth in milk at 37 ° C. 8. A composition that is suitable as a starter culture for the production of a dairy product, comprising a strain of lactic acid bacteria according to any one of claims 4 to 7, or a combination thereof. 9. The composition according to claim 8, including: ) a strain belonging to the species ZTsYusssch 1ygtorY1 and 8 according to claim 6 or 7; and and) a strain belonging to the species BasUnus béichex (subspecies b1dapsi8) according to claim 4 or 5. 10. Composition according to any one of paragraphs.8 and 9, which is in frozen, lyophilized or liquid form. 11. Method for the production of dairy product, which includes the fermentation of the dairy substrate with a strain of lactic acid bacteria according to any one of claims 4 to 7 or a composition according to any one of claims 8 and 9. 12. Dairy product comprising a strain of lactic acid bacteria according to any one of claims 4 to 7. 13. The use of a strain of lactic acid bacteria according to any one of claims 4 to 7 for the production of dairy product. 14. The use of a texturing strain of lactic acid bacteria according to any one of claims 4 to 7 to increase the texture of the dairy product. - 10 029862